Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure

Butler, Max B.; Short, Nina E.; Maniou, Eirini; Alexandre, Paula; Greene, Nicholas D. E.; Copp, Andrew J.; Galea, Gabriel L.

doi:10.1242/jcs.230300

Cited by 29 publications

(65 citation statements)

References 61 publications

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“…Neuroepithelial apical constriction has been documented in the mouse by ourselves and others 12,21,38,39 .…”

Section: Introductionmentioning

confidence: 85%

“…In both mouse and chick embryos, neural fold elevation requires apical tension generated by actomyosindependent apical constriction 21,25 and variable apical areas in Cre;Fl/-embryos suggest differential constriction within the mosaic neuroepithelium. Cellular mechanical tension is commonly inferred from recoil of cell borders immediately following laser ablation.…”

Section: Mosaic Vangl2 Deletion Diminishes Neuroepithelial Apical Conmentioning

confidence: 99%

“…Cellular mechanical tension is commonly inferred from recoil of cell borders immediately following laser ablation. We previously demonstrated that annular laser ablations in the apical neuroepithelium produce actomyosin-dependant, rapid initial shrinkage of the cluster of cells within the annulus as they are untethered from the surrounding tissue 21 . Here, the reduction in apical area of cells or cell clusters following ablation will be referred to as "retraction" to differentiate it from spontaneous apical "constriction" during live imaging.…”

Section: Mosaic Vangl2 Deletion Diminishes Neuroepithelial Apical Conmentioning

confidence: 99%

“…Consequently, mislocalisation of PCP components in individual mutant cells necessarily prevents their neighbours from correctly localising the opposing complex components [18][19][20] . Protein-level planar-polarised orientation of Vangl2 is not apparent in the late PNP of the mouse embryo, where pseudostrati ed neuroepithelial cells have complex apical shapes often without predictably oriented borders 13,21 . Nonetheless, planar-polarised properties do emerge in this complex epithelium, including preferential arrangement of cell apices and supra-cellular actomyosin pro les in a mediolateral orientation 13,21 .…”

Section: Introductionmentioning

confidence: 99%

“…The neuroepithelium has a predominantly apical localisation of F-actin and non-muscle myosin-II 40,41 . Pharmacological inhibition of myosin activation reduces apical neuroepithelial mechanical tension and causes PNP widening 21 . The molecular regulation of apical constriction has been extensively studied in non-mammalian vertebrates, demonstrating cell-autonomous basolateral-to-apical shuttling of myosin onto an apical F-actin network 23,29,42 .…”

Section: Introductionmentioning

confidence: 99%

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Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion

Galea

Maniou

Marshall

et al. 2020

Preprint

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Post-zygotic mutations that generate tissue mosaicism are increasingly associated with severe congenital defects, including those arising from failed neural tube closure. We observed that elevation of the neural folds during mouse spinal neurulation is vulnerable to deletion of the planar cell polarity core component Van Gogh-like (Vangl)2 in as few as 16% of neuroepithelial cells. Vangl2-deleted cells are typically dispersed throughout the neuroepithelium, and each non-autonomously prevents apical constriction by an average of five Vangl2-replete neighbours. This inhibition of apical constriction involves reduced myosin-II recruitment to neighbour cell borders and shortening of basally-extending microtubule tails, which are known to facilitate apical constriction. Vangl2-deleted cells themselves continue to apically constrict and preferentially recruit myosin-II to their apical cell cortex rather than to apical cap sarcomere-like organisations. Such non-autonomous effects can explain how post-zygotic mutations affecting a minority of cells can cause catastrophic failure of morphogenesis leading to clinically important birth defects.

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“…Neuroepithelial apical constriction has been documented in the mouse by ourselves and others 12,21,38,39 .…”

Section: Introductionmentioning

confidence: 85%

Section: Mosaic Vangl2 Deletion Diminishes Neuroepithelial Apical Conmentioning

confidence: 99%

Section: Mosaic Vangl2 Deletion Diminishes Neuroepithelial Apical Conmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion

Galea

Maniou

Marshall

et al. 2020

Preprint

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Refinement of inducible gene deletion in embryos of pregnant mice

Savery¹,

Maniou²,

Culshaw³

et al. 2019

Birth Defects Research

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CreERT2‐mediated gene recombination is widely applied in developmental biology research. Activation of CreERT2 is typically achieved by injection of tamoxifen in an oily vehicle into the peritoneal cavity of mid‐gestation pregnant mice. This can be technically challenging and adversely impacts welfare. Here we characterize three refinements to this technique: Pipette feeding (not gavage) of tamoxifen, ex vivo CreERT2 activation in whole embryo culture and injection of cell‐permeable TAT‐Cre into Cre‐negative cultured embryos. We demonstrate that pipette feeding of tamoxifen solution to the mother on various days of gestation reliably activates embryonic CreERT2, illustrated here using β‐Actin CreERT2, Sox2 CreERT2, T CreERT2, and Nkx1.2 CreERT2. Pipette feeding of tamoxifen induces dose‐dependent recombination of Rosa26 mTmG reporters when administered at E8.5. Activation of two neuromesodermal progenitor‐targeting Cre drivers, T CreERT2, and Nkx1.2 CreERT2, produces comparable neuroepithelial lineage tracing. Dose‐dependent CreERT2 activation can also be achieved by brief exposure to 4OH‐tamoxifen in whole embryo culture, allowing temporal control of gene deletion and eliminating the need to treat pregnant mice. Rosa26 mTmG reporter recombination can also be achieved regionally by injecting TAT‐Cre into embryonic tissues at the start of culture. This allows greater spatial control over Cre activation than can typically be achieved with endogenous CreERT2, for example by injecting TAT‐Cre on one side of the midline. We hope that our description and application of these techniques will stimulate refinement of experimental methods involving CreERT2 activation for gene deletion and lineage tracing studies. Improved temporal (ex vivo treatment) and spatial (TAT‐Cre injection) control of recombination will also allow previously intractable questions to be addressed.

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Two-Photon Cell and Tissue Level Laser Ablation Methods to Study Morphogenetic Biomechanics

Marshall¹,

Maniou²,

Moulding³

et al. 2022

Methods in Molecular Biology

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Laser ablation is routinely performed to infer mechanical tension in cells and tissues. Here we describe our method of two-photon laser ablation at the cellular and tissue level in mouse embryos. The primary outcome of these experiments is initial retraction following ablation, which correlates with, and so can be taken as a measure of, the tensile stress that structure was under before ablation. Several experimental variables can affect interpretation of ablation tests.Pre-test factors include differences in physical properties such as viscoelasticity between experimental conditions. Factors relevant during the test include viability of the cells at the point of ablation, image acquisition rate and the potential for over-zealous ablations to cause air bubbles through heat dissipation. Post-test factors include intensity-biased image registration that can artificially produce apparent directionality. Applied to the closing portion of the mouse spinal neural tube, these methods have demonstrated long-range biomechanical coupling of the embryonic structure and have identified highly contractile cell populations involved in its closure process.

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Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure

Cited by 29 publications

References 61 publications

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion

Refinement of inducible gene deletion in embryos of pregnant mice

Two-Photon Cell and Tissue Level Laser Ablation Methods to Study Morphogenetic Biomechanics

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